We report the molecular and functional characterization of murine Slc26a6, the putative apical chloride-formate exchanger of the proximal tubule. The Slc26a6 transcript is expressed in several tissues, including kidney. Alternative splicing of the second exon generates two distinct isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion of a 23-residue NH2-terminal extension. Functional comparison with murine Slc26a1, the basolateral oxalate exchanger of the proximal tubule, reveals a number of intriguing differences. Whereas Slc26a6 is capable of Cl−, SO , formate, and oxalate uptake when expressed in Xenopus laevis oocytes, Slc26a1 transports only SO and oxalate. Measurement of intracellular pH during the removal of extracellular Cl− in the presence and absence of HCO indicates that Slc26a6 functions as both a Cl−/HCO and a Cl−/OH− exchanger; simultaneous membrane hyperpolarization during these experimental maneuvers reveals that HCO and OH− transport mediated by Slc26a6 is electrogenic. Cis-inhibition and efflux experiments indicate that Slc26a6 can mediate the exchange of both Cl− and SO with a number of substrates, including formate and oxalate. In contrast, SO and oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate. The data indicate that Slc26a6 encodes an apical Cl−/formate/oxalate and Cl−/base exchanger and reveal significant mechanistic differences between apical and basolateral oxalate exchangers of the proximal tubule.
- proximal tubule
- anion exchange
anion exchange at the plasma membrane is primarily mediated by the products of two structurally distinct gene families; the anion exchanger (AE) genes, which form a subset of the bicarbonate transporter, SLC4superfamily (41, 52), and the SLC26 or “sulfate permease” gene family (10, 33). The mammalianSLC26 gene family has emerged over the last seven years through a combination of expression cloning (4), subtractive cDNA cloning (61), and positional characterization of human disease genes (10). Evolving physiological roles for specific family members include transepithelial salt transport (10, 45), thyroidal iodide transport (46), development and function of the inner ear (10,61), sulfation of extracellular matrix (43), and the renal excretion of both bicarbonate (42) and oxalate (17). The various substrates transported by the SLC26 anion exchangers include sulfate (SO ), chloride (Cl−), iodide (I−), formate, oxalate, hydroxyl ion (OH−), and bicarbonate (HCO ) (4, 17, 36, 43, 45, 49).
The SLC26 gene family is highly conserved across evolution, with identifiable homologues in bacteria, fungi, yeast, plants, and animals (10). The Drosophila genome contains at least nine family members, and the existence of new mammalian paralogs has been suspected for some time (10). The cloning of several of these new genes has been recently reported (19, 30, 31, 53,54), and the family appears to encompass 10 members and 1 pseudogene (Mount DB, unpublished observations). Physiological interest in the characterization of novel family members was stimulated in particular by the observation that SLC26A4 can function as a Cl−/formate exchanger (45), because Cl−/formate exchange is thought to play a pivotal role in the transepithelial transport of NaCl by the renal proximal tubule (56, 57). However, Slc26a4 is not expressed in the proximal tubule but is instead found at the apical membrane of β-intercalated cells, where it appears to play a role in renal bicarbonate excretion (42). The sixth member of the gene family, SLC26A6, was recently cloned in both humans (SLC26A6) (30, 54) and mice (Slc26a6, also known as CFEX) (19). Functional characterization of Slc26a6 indicates that it can mediate both Cl−/formate and Cl−/Cl− exchange (19), whereas the human ortholog was nonfunctional (54). The immunolocalization of SLC26A6 and Slc26a6 indicates expression at the apical membrane of epithelial cells (19, 30) and suggests that this gene encodes the proximal tubule Cl−/formate exchanger. Important unresolved issues include whether SLC26A6 also mediates Cl−/oxalate exchange and Cl−/base exchange (Cl−/OH−and/or Cl−/HCO exchange). Apical oxalate transport by a DIDS-sensitive anion exchanger is thus thought to play an important role in oxalate secretion by the proximal tubule, in concert with basolateral oxalate transport mediated by Sat-1 (SLC26A1/Slc26a1). There is also a significant body of evidence suggesting that apical Cl−/base exchange functions in transepithelial NaCl absorption by the proximal tubule (24). Moreover, the ability of SLC26 proteins to function as Cl−/base exchangers identifies Slc26a6 as a candidate for both the apical Cl−/base exchanger of the proximal tubule and the apical CFTR-dependent bicarbonate transporter(s) in the lung (26), submandibular gland (27), and exocrine pancreas (7, 27). We report the initial exploration of these issues, in addition to a functional comparison of the murine Slc26a1 and Slc26a6 anion exchangers.
Molecular characterization of Slc26a6 and Slc26a1.
Human SLC26A6 exons were initially identified in the draft sequences of the BAC clone RP11–148G20 and the PAC clone RP4–751E10, using tblastn searches of the HTGS database with the SLC26A1–4 proteins. A blastn search of the mouse expressed sequence tag (EST) database using the extracted human exon contig yielded a Sugano mouse IMAGE clone (2076921), with 5′- and 3′-EST entries that exhibited modest homology to the NH2and COOH termini of known SLC26 proteins. This full-length cDNA was obtained from Research Genetics (Birmingham, AL), sequenced on both strands using fluorescent dye terminator chemistry (Applied Biosystems), and submitted to GenBank (AF248494, 3/23/00). Ablastn search of the Celera mouse genomic database yielded a 500-kb contig containing the 11-kb Slc26a6 gene, and a subsequent blastn search of the mouse EST database using a 1.7-kb region between the start of the 2076921 EST and the 3′-untranslated region (UTR) of the upstream gene yielded three RIKEN 5′-ESTs that overlap with 2076921 but have a different 5′-end. This alternative 5′-end was cloned by RT-PCR from mouse intestine total RNA, using a sense primer in exon 1a (5′-TACACGAGTTACCCTCTGAGG-3′) and an antisense primer from within exon 4 (5′-TACAGACCAAACATAGGAGGC-3′), as described (37). The two amplified PCR fragments obtained (see Fig. 2) were subcloned into pCR2.1 (Invitrogen) and sequenced. Finally, for the purpose of functional comparison to Slc26a6, we identified and sequenced a full-length mouse Slc26a1 EST (IMAGE clone 1450460).
The analysis of nucleotide and amino acid sequence utilized Vector NTI 6.0 (Informax), supplemented by GRAIL (http://compbio.ornl.gov/Grail-1.3/), Phosphobase (http://www.cbs. dtu.dk/databases/PhosphoBase/), MattInspector (http://transfac. gbf.de/cgi-bin/matSearch/matsearch.pl), TESS (http://www. cbil.upenn.edu/cgi-bin/tess/tess33?RQ=SEA-FR-Query), and Prosite (http://www.expasy.ch/prosite/). Genomic localization ofSlc26a1 and Slc26a6 exploited Celera genomic contigs encompassing the two genes, the UniSTS website (NCBI), and the Mouse Genome Database (MGD;http://www. informatics.jax.org/mgihome/).
Northern blot analysis.
RNA was extracted from C57BL/6J mice using guanidine isothiocyanate and cesium chloride. Total RNA (10 μg/lane) was size-fractionated by electrophoresis (5% formaldehyde, 1% agarose), transferred to a nylon membrane (Stratagene), and probed sequentially with32P-labeled randomly primed probes corresponding to full-length GAPDH and a 3′-probe from Slc26a6 (nucleotides 2339–2673 of Slc26a6b). Hybridization was performed overnight at 42°C in Express-Hyb solution (Clontech), and membranes were washed twice for 10 min at room temperature in 2× SSCP/0.1% SDS and twice for 1 h at 65°C in 0.1× SSCP/0.1% SDS.
Expression of Slc26a1 and Slc26a6 in Xenopus laevis oocytes.
The entire inserts of the Slc26a6b and Slc26a1 cDNAs were transferred to the pGEMHE X. laevis expression vector (29) using EcoRI and XbaI. The Slc26a6b and Slc26a1 expression constructs were linearized using NheI andNotI, respectively, and cRNA was transcribed in vitro using T7 RNA polymerase and mMESSAGE mMACHINE kits (Ambion). Defolliculated oocytes were injected with 25–50 nl of water or a solution containing cRNA at a concentration of 0.5 μg/μl (12.5–25 ng/oocyte), using a Nanoliter-2000 injector (WPI Instruments, Sarasota, FL). Oocytes were incubated at 17°C in 50% Leibovitz's L-15 media supplemented with Pen-Strep (1,000 U/ml) and glutamine for 2–3 days before uptake assays.
For sulfate uptakes, oocytes were preincubated for 20 min in chloride-free uptake medium [100 mMN-methyl-d-glucamine (NMDG)-gluconate, 2 mM potassium gluconate, 1 mM calcium gluconate, 1 mM magnesium gluconate, 10 mM HEPES-Tris, pH 6.0 or pH 7.5, as indicated], followed by a 60-min uptake in the same medium with 1 mM K2 35SO4. The cells were then washed three times in uptake buffer with 5 mM nonradioactive K2SO4 to remove tracer activity in the extracellular fluid. The oocytes were dissolved individually in 10% SDS, and tracer activity was determined by scintillation counting. Cl−, formate, and oxalate uptakes were assayed using the same Cl−-free uptake solutions, substituting 8.3 mM36Cl−, 500 μM [14C]oxalate, or 50 μM [14C]formate for labeled sulfate. For sulfate exchange (see Fig. 5 C) and cis-inhibition (Fig.5 A), the concentration of NMDG-gluconate in the uptake solution was adjusted to maintain isotonic osmolality, which was experimentally confirmed using a Fiske 110 osmometer.Cis-inhibition experiments used the Na+ salts of the relevant anions. All radioisotopes were from New England Nuclear (Boston, MA).
The uptake experiments all included 12–18 oocytes in each experimental group, statistical significance was defined as two-tailed (P < 0.05), and results are reported as means ± SE.
Oocytes were studied 3–11 days postinjection. The CO2/HCO -free ND96 contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES (pH 7.5 and 195–200 mosmol/kgH2O) and bubbled with 100% O2 during experiments. For CO2/HCO -equilibrated solutions, 33 mM NaHCO3 replaced 33 mM NaCl. In 0-Na+ solutions, choline replaced Na+. In 0-Cl− solutions, gluconate replaced Cl−. All solutions were titrated to pH 7.5 and continuously bubbled with CO2-balanced O2 to maintain Pco 2 and pH. Ion-selective microelectrodes were prepared and calibrated, and experiments were performed as previously described for NBC (40) and NDAE1 (41). All pH electrodes had slopes of at least −56 mV/decade concentration change. Where statistical tests were indicated, a two-tailed Student'st-test assuming unequal variances was used. Pvalues <0.05 were considered statistically significant.
Sequence comparison indicates that the murine Slc26a6 and human SLC26A6 orthologs share only 78% identity at the amino acid level, much lower than the median of 86% for human and mouse orthologous pairs (32). However, the murine and human genes are clearly orthologous, such that both are flanked at the 5′-ends by theFMI-1/MEGF-2 gene and at the 3′-end by the UQCRC1and ColA7 genes (13, 28). The gene and sequence tagged site content of these contigs confirm that the humanSLC26A6 gene is on chromosome 3p21 (30) and the murine gene is in the syntenic segment of chromosome 9, at ∼61 cM. In particular, the murine ColA7 gene is physically linked ∼40 kb 3′ of Slc26a6 and is known to be localized on mouse chromosome 9 at 61.0 cM (UniSTS marker 859381) (28).
The Slc26a6 and SLC26A6 genes share a similar organization, encompassing 21 coding exons and ∼10 kb of genomic DNA. Both genes include an alternative 5′ noncoding exon (exon 1b, 945 nucleotides 5′ of coding exon 2 in Slc26a6). Nonquantitative RT-PCR using primers in exons 1a and 4 (see Fig. 2 A) suggests that the isoform in which exon 1b has been spliced out, denoted Slc26a6a, is expressed at a lower level than Slc26a6b; our functional experiments utilized a Slc26a6b construct. The identity of the two RT-PCR fragments (see Fig. 2 A) was verified by subcloning and sequencing of the amplified bands. The intron-exon boundaries in Slc26a6were determined by comparison of the various Slc26a6 cDNAs to the murine Celera contig, and all of the donor and acceptor sites were found to conform to consensus splice sites (39) (Table1). The inclusion of exon 1b in the longer Slc26a6b transcript results in a frame shift and a start codon within exon 2. The predicted Slc26a6b protein is thus 23 amino acids shorter than Slc26a6a (Fig. 1). The start codons in exon 1a and exon 2 are both predicted Kozak sites (21), with purines at position −3 and G at position +4. However, it is conceivable that the Kozak site in exon 2 is in fact preferred for translation initiation in both the Slc26a6a and Slc26a6b transcripts; formal proof that the start codon in exon 1a is utilized will require the generation of an antibody to the putative NH2-terminal extension of the Slc26a6a protein. Slc26a6b is essentially identical to the sequence of mouse “CFEX” (19), except for codons 2 (glutamate in Slc26a6b, glycine in CFEX), 65 (valine in Slc26a6b, indeterminate in CFEX), and 549 (proline in Slc26a6b, arginine in CFEX). Exon 1b is conserved in the human genomic sequence; the cDNA reported by Waldegger et al. (54) corresponds to SLC26A6a, whereas that reported by Lohi et al. (30) corresponds to SLC26A6b. The functional significance of the alternative NH2 terminus in the Slc26a6a isoform is not yet known; however, note is made of the conservation of the sequence, TQALLS, in mice, humans, and pigs (Fig. 1 and data not shown, porcine SLC26A6a). The murine Slc26a6b isoform lacking the NH2-terminal extension is functional (see Figs. 4-7); hence, this sequence is not required for transport activity.
Northern blot analysis indicates that the Slc26a6 gene is widely expressed, with a 3.0-kb transcript detected in intestine, kidney, testis, brain, muscle, heart, and stomach (Fig.2 B); hybridization levels for GAPDH were approximately equal (not shown). The widespread expression of Slc26a6 is consistent with the presence of a CpG island overlapping exon 1a in the Celera genomic contig (Fig.3), conserved in the human gene (data not shown). Although the transcriptional start site has not been mapped for either species, the most 5′ Slc26a6a ESTs begin ∼100 bp 5′ of the start codon in exon 1a. The genomic DNA flanking in exon 1a suggests a TATA-less promoter, rich in predicted Sp1 sites (Fig. 3).
For the purpose of functional comparison to Slc26a6, we also characterized mouse Slc26a1 (Sat-1), identified on a full-length EST cDNA (Fig. 1 B). The predicted Slc26a1 protein is 91 and 76% identical to the rat and human SLC26A1 proteins, respectively. As is the case with Slc26a6/SLC26A6, the mouse and human Slc26a1/SLC26A1 genes are clearly orthologous. Large genomic contigs containing the mouse (Celera) and human (GenBank NT_006111) genes reveal a conserved organization, such that both are flanked at the 5′-end by the FGFRL-1(59), GAK (18), andDAGK4 (9) genes and at the 3′-end by the l-iduronidase gene. The murineFGFRL-1 and l-iduronidase genes have both been localized on mouse chromosome 5 at ∼57 cM (59), syntenic with the region of human chromosome 4p16 containing SLC26A1, GAK (18), andDAGK4 (9). The genomic organization of the twoSLC26A1/Slc26a1 genes is also conserved, although analysis of a number of 5′ Slc26a1 ESTs reveals the existence of two 5′ noncoding exons in the murine gene (Table2). The more 3′ of these two noncoding exons, denoted exon 1b, is excluded from a number of ESTs, indicating that it is alternatively spliced. Of note, the relative position of the junction between the two coding exons ofSLC26A1/Slc26a1 and SLC26A2 (DTDST), which together form a separate branch of the gene family, is conserved in the respective mouse and human genes.
Anion transport in X. laevis oocytes.
We have characterized the physiological properties of mouse Slc26a6b and Slc26a1 in X. laevis oocytes using similar protocols to those published for SLC26A1 (4, 25, 43), SLC26A2 (43), SLC26A3 (36), and SLC26A4 (45,46).
Figure 4 illustrates the uptake of35SO and 36Cl−by oocytes injected with Slc26a1 and Slc26a6, as a function of extracellular pH and extracellular Cl− or SO . As shown in Fig. 4 A, Slc26a1 mediates Cl−- and Na+-independent sulfate transport (148 ± 9 and 237 ± 37 pmol · oocyte−1 · h−1 at pH 7.4 and 6.0, respectively, vs. 2.0 ± 0.2 and 5.8 ± 0.9 pmol · oocyte−1 · h−1 in water-injected controls), as does Slc26a6 (605 ± 40 and 625 ± 53 pmol · oocyte−1 · h−1at pH 7.4 and 6.0, respectively). The difference in35SO uptake between pH 7.4 and 6.0 is significant for Slc26a1 (P < 0.02) but not for Slc26a6. Both transporters are more sensitive to 1 mM DIDS at the lower pH (301 ± 24 pmol · oocyte−1 · h−1 at pH 7.4 vs. 101 ± 10 pmol · oocyte−1 · h−1 at pH 6.0 for Slc26a6 in the presence of DIDS). In contrast, Cl−uptake in Slc26a6/CFEX-expressing oocytes was reported by Knauf et al. (19) to be highly sensitive to 100 μM DIDS, with no significant difference between pH 7.4 and 6.5; this higher sensitivity may have masked an effect of extracellular pH. It is also likely that DIDS sensitivities of Slc26a6 and other members of the family are highly dependent on both the concentration and the identity of the transported anion (16), although we note that SLC26A3 and SLC26A4 are also only modestly sensitive to DIDS (1, 46).
Oocytes expressing Slc26a6 transported 36Cl−(Fig. 4 B), although, again, there was no consistent difference between pH 7.4 and 6.0 for Slc26a6-injected groups (4,345 ± 243 and 4,193 ± 109 pmol · oocyte−1 · h−1 at pH 7.4 and 6.0, respectively, vs. 106 ± 12 and 99 ± 16 pmol · oocyte−1 · h−1 in water-injected controls). In contrast to Slc26a6, Slc26a1-injected oocytes did not take up 36Cl−; this is in agreement with transport studies using basolateral membrane vesicles from renal cortex, which indicate that the basolateral SO /HCO exchanger in these preparations does not transport Cl− (23). We considered that Slc26a1 might require the presence of extracellular SO to transport Cl−; however, Slc26a1 mediates minimal Cl− uptake in either the presence or the absence of 25 mM SO (Fig. 4 B).
The absolute rates of 35SO uptake for Slc26a1-injected oocytes shown in Fig. 4 A are reproducibly lower than those of Slc26a6-injected oocytes. However, SO uptake rates for Slc26a1 are much higher in the presence of extracellular Cl−, closer to the values for Slc26a6 in the absence of Cl− (Fig. 4 C). In contrast, 35SO uptake by Slc26a6-injected cells is strongly cis-inhibited by extracellular Cl− (Fig. 4 C), consistent with the ability of this exchanger to mediate 36Cl−uptake (Fig. 4 B). The increase in35SO transport by Slc26a1 at an extracellular pH of 6.0 is greater in the presence (Fig.4 D) than in the absence (Fig. 4 A) of extracellular Cl−.
A shared property of the SLC26 anion exchangers iscis-inhibition of the uptake of a given ion by other substrates (19, 36, 43, 45). To assess the repertoire of substrates capable of cis-inhibiting Cl− uptake by Slc26a6, we incubated Slc26a6-injected oocytes with sulfate, formate, halides, nitrate, and lactate; all but lactate significantly inhibit 36Cl− uptake (Fig.5 A). A similar profile was obtained for SO transport (Fig. 5 B). We also assessed the ability of Slc26a6 to mediate SO exchange by measuring efflux of this tracer in the presence of extracellular substrates (45). For this experiment, oocytes were incubated in 35SO uptake media for 1 h and then incubated in the absence (control group) or the presence of the substrates indicated in Fig. 5 C to see whether these anions would transstimulate efflux of35SO over a 30-min period. Because SLC26A4 is known to transport formate and Cl−, but neither oxalate (45) nor SO (46), we reasoned that Slc26a6 might not catalyze formate/SO exchange, such that extracellular formate would not stimulate efflux of 35SO . However, Fig. 5 Cindicates that Slc26a6 mediates the exchange of SO with SO , Cl−, formate, and oxalate; the residual 35SO content of the relevant oocyte groups is substantially lower than that of the control group, indicating loss of 35SO due to exchange with the extracellular anions. Some efflux of35SO likely occurred in the control oocytes during the 30-min post-uptake period, because the picomoles remaining in this group (331 ± 18 pmol/oocyte) are approximately half that of the oocytes after the 1-h uptakes shown in Fig.4 A.
As one would predict from the cis-inhibition experiments shown in Fig. 5, Slc26a6-injected oocytes transport both oxalate (487 ± 50 vs. 12 ± 1 pmol · oocyte−1 · h−1 in water-injected controls, Fig.6 A) and formate (45 ± 5 vs. 7 pmol · oocyte−1 · h−1in water-injected controls, Fig. 6 B). In contrast, Slc26a1 mediates the transport of oxalate (72 ± 2 pmol · oocyte−1 · h−1, Fig.6 A), as reported for rat Slc26a1 (17), but not formate (7 pmol · oocyte−1 · h−1, Fig.6 B).
The activation of (rat) Slc26a1 by extracellular Cl− (Fig.4 C) has been observed before (43) but not explored in detail. To extend this observation, we first examined the effect of several monovalent ions (halides, formate, and lactate) on35SO uptake by Slc26a1-injected oocytes (Fig. 7 A). Whereas the divalent substrates SO and oxalate are stronglycis-inhibitory for Slc26a135SO uptake (Fig. 5, A andB), monovalent ions share activation of35SO uptake with Cl− (Fig.7 A). Although the ability of Slc26a1 to transport all of these monovalent ions has not been examined, it transports neither Cl− (Fig. 4 B) nor formate (Fig. 6 B). Moreover, I− and Br− are bothcis-inhibitory for both SO and Cl− uptake via Slc26a6, indicating that they are potential substrates, as shown directly for SLC26A4 in the case of I− (46); by extension, these are not likely substrates for Slc26a1. The activation of Slc26a1 by impermeant anions is not unique to SO transport, because oxalate transport in Slc26a1-injected oocytes is also higher in the presence of Cl− and other anions (Fig. 7 B). Slc26a6 serves as a control for the latter experiment, in that oxalate transport by oocytes injected with this cRNA is strongly cis-inhibited by these anions (Fig. 7 B).
Oocyte intracellular pH and electrophysiology.
To determine whether Slc26a6 functions as a Cl−/HCO exchanger, measurements were made of intracellular pH (pHi) in response to the manipulation of bath HCO and Cl−. The initial addition of CO2/HCO to the bath solution results in the acidification of oocytes due to CO2 plasma membrane diffusion, then intracellular hydration and dissociation, forming intracellular H+ and HCO . Figure 8illustrates an experiment with single water- and Slc26a6-injected oocytes; however, these results were observed for Slc26a6-injected oocytes from five separate frogs; rates are reported for both the single oocytes shown in Fig. 8 and for the mean of several oocytes (in parentheses). Figure 8 shows that a water-injected oocyte exposed to 5% CO2/33 mM HCO (pH 7.5) acidifies by 0.44 pH units (−0.46 ± 0.01, n= 8) at an initial rate of 46.0 × 10−4 pH units/s (−382 ± 19 × 10−5 pH units/s,n = 8). The initial pHi of Slc26a6 oocytes is essentially the same as that of water-injected control (water-injected, 7.26 ± 0.03, n = 8; Slc26a6, 7.29 ± 0.03, n = 10); addition of 5% CO2/33 mM HCO causes a fall in pHi of 0.50 pH units (−0.46 ± 0.02,n = 10) at an initial rate of 35.0 × 10−4 pH units/s (−387 ± 15 × 10−5 pH units/s, n = 10). Slc26a6 oocytes are depolarized (−26.3 ± 4.5 mV, n = 10) compared with control oocytes (−44.8 ± 4.3 mV, n= 8). The addition of HCO causes a slight but abrupt depolarization in Slc26a6-injected oocytes (3.1 ± 0.6 mV,n = 9), reminiscent of the anion conductance observed in oocytes expressing NDAE1 (41). Cl−replacement (gluconate) does not affect pHi of the water-injected control (+6.0 ± 2.2 × 10−5 pH units/s, n = 8; Fig. 8 A). However, Fig.8 B illustrates that Cl− removal increases pHi of a Slc26a6 oocyte at the rate of 44 × 10−5 pH units/s (+72 ± 8.8 × 10−5pH units/s, n = 10; Fig. 8 B), which ceases after Cl− readdition. Surprisingly, this gluconate replacement evokes a 37-mV hyperpolarization (−22.7 ± 2.9 mV,n = 9; vs. +0.2 ± 2.0 mV, n = 8 for controls). A second Cl− removal in Fig. 8 Balkalinizes the cell at 28 × 10−5 pH units/s (+41 ± 6.2 × 10−5 pH units/s,n = 8) and reproduces the hyperpolarization (−18.6 ± 3.8 mV, n = 8). In all the experiments with Slc26a6-injected oocytes, the second alkalinization induced by Cl− removal, which occurs at a higher pHi, has a lower rate (+72 × 10−5 pH units/s for the first alkalinization and +41 × 10−5 vs. +6.0 × 10−5 pH units/s for the single Cl− removal in water-injected oocytes).
We also replaced Na+ with choline to test cation dependence of Slc26a6. Na+ removal and replacement did not obviously affect pHi. Before CO2 removal, pHirose to 7.2, i.e., almost the non-HCO level. Finally, removal of 5% CO2/33 mM HCO elicits a robust alkalinization and pHi overshoot to 7.9 (7.83 ± 0.07, n = 10), indicative of cellular HCO loading [change in (Δ)pHi for Slc26a6 is +0.53 ± 0.07, n = 10] (40,41). This overshoot is not observed in controls (ΔpHi for controls is +0.02 ± 0.04,n = 8) (40, 41).
Because Slc26a6 clearly functions as a Cl−/HCO exchanger (Fig. 8 B), we tested whether it could function as a Cl−/OH− exchanger. For these experiments, we continuously bubbled all the non-HCO solutions with 100% O2 as previously described in the characterization of NDAE1 (41). Figure 9illustrates the non-HCO responses of one control and one Slc26a6 oocyte; the data for these two oocytes are detailed in the legend for Fig. 9. Data for the mean of several oocytes and several frogs are written in parentheses. Bath Cl− removal of control oocytes (Fig. 9 A) does not change pHi(−2.1 ± 1.8 × 10−5 pH units/s,n = 7) or membrane potential (+1.1 ± 1.7 mV,n = 14). Nevertheless, this same maneuver alkalinized (+27 ± 6.4 × 10−5 pH units/s,n = 6) and hyperpolarized (−9.1 ± 3.0 mV,n = 11; P < 0.008) Slc26a6 oocytes (Fig. 9 B). Cl− readdition to the bath stopped the alkalinization and returned membrane potential to the initial value. However, the readdition of Cl− evoked a large transient depolarization (+39.6 ± 4.9 mV, n = 11 for Slc26a6 vs. −4.6 ± 0.9 mV, n = 14 for controls; P < 0.000002) followed by a smaller sustained depolarization.
We report here the molecular and functional characterization of Slc26a1 and Slc26a6, the first and sixth members of the murine Slc26 gene family of anion exchangers. We have also reported on the chromosomal localization and genomic structure of the two genes, along with evidence of alternative splicing of both transcripts. Immunolocalization and functional characterization recently identified Slc26a6 as the putative Cl−/formate exchanger of the renal proximal tubular epithelium (19), and our data indicate that Slc26a6 can also mediate SO exchange, Cl−/oxalate, Cl−/OH−, and Cl−/HCO exchange. Basolateral oxalate-SO /HCO exchange in the proximal tubule is thought to be mediated by Slc26a1/SLC26A1 (Sat-1) (17), and a direct functional comparison indicates that SO and oxalate exchange by these homologous transporters is mechanistically distinct.
The sequence comparison of mouse Slc26a6 reveals that the closest characterized relative is prestin (Slc26a5), with 39% identity at the amino acid level, whereas Slc26a1 is closest to Slc26a2 (DTDST, 47% identity). The COOH-terminal domains of both Slc26a1 and Slc26a6 contain predicted sulfate transporter and anti-sigma (STAS) domains, recently defined by virtue of homology between the SLC26gene family and bacterial anti-sigma factor antagonists (2). Structural predictions suggest a role for the STAS domain in nucleotide binding and/or hydrolysis (2). With the exception of a COOH-terminal type I PDZ interaction motif (51) (see below), analysis of the Slc26a6 protein with motif-based algorithms (44, 60) does not reveal other protein-signaling domains or motifs. Mouse and rat Slc26a1 (4) predict a COOH-terminal type I PDZ binding motif (S-A-L); however, this motif is not conserved in the human protein (GenBank AF297659). Analysis using both the Prosite and Phosphobase (22) databases reveals that Slc26a1 and Slc26a6 are both potential substrates for a number of protein kinases, including tyrosine kinases, protein kinase A, and protein kinase C (Fig. 1).
We utilized heterologous expression in X. laevis oocytes for the functional characterization of Slc26a1 and Slc26a6. Isotopic flux studies indicate that Slc26a6 is a versatile anion exchanger, capable of transporting SO , Cl−, formate, and oxalate, whereas Slc26a1 transports only SO and oxalate in flux assays. The direct measurement of changes in pHi elicited by the removal of extracellular Cl− in both the presence and absence of HCO indicates that Slc26a6 functions as a Cl−/HCO and Cl−/OH− exchanger (Figs. 8 and 9). While this manuscript was in review, Wang et al. (58) reported that Slc26a6-injected X. laevis oocytes can mediate Cl−/HCO exchange. The lack of stimulation of 36Cl− uptake by Slc26a6 by a more acidic extracellular medium (Fig. 4 B) is somewhat surprising, given that this protein can mediate Cl−/OH− exchange (Fig. 9). However, similar findings for the pH dependency of Slc26a6 Cl− uptake were reported by Knauf et al. (19).
Slc26a6 shares the ability to mediate Cl−/base exchange with Slc26a3 (34) and Slc26a4 (42, 49). However, the electrophysiology of this mode of anion transport has not been studied in the SLC26 anion exchangers. We have found that removal of extracellular Cl− in the presence of HCO evokes a significant hyperpolarization. The simplest explanation is that Slc26a6 mediates electrogenic HCO transport. That is, these data are consistent with 1) cation (e.g., H+) coexit with Cl−; 2) the entry of another anion with HCO into the cell; or 3) exchange of more than one HCO ion for each Cl− ion that exits. We cannot rule out the possibility that Slc26a6 overexpression somehow evokes an otherwise silent conductance natively present in the oocyte, and future experiments will examine the nature of these Cl−-suppressed voltage or current changes. However, unmasking an otherwise silent conductance in response to Slc26a6 expression seems unlikely, because there is not a precedent in the literature for a “Cl−-inhibited anion current” as measured in our experiments.
Although complete functional characterization is lacking for many of the SLC26 anion exchangers, the range of substrates intrinsic to Slc26a6 is tentatively only matched by that of SLC26A3 (DRA) (5,34, 36, 54). Of note, however, formate transport has not been reported for SLC26A3, and only low-level oxalate, Cl−, and SO uptake compared with water-injected controls has been reported for SLC26A3-injected oocytes (36, 48). In contrast to Slc26a6, Slc26a1 does not transport Cl− or formate but does transport SO and oxalate (Figs. 4-7). SLC26A2, in turn, is known to transport monovalent anions such as Cl−, I−, and formate, but not divalent anions such as SO and oxalate (45, 46). This combination of functional divergence and structural homology within the SLC26 gene family will no doubt aid the structure-function analysis of anion specificity.
The data reported here reveal that transport of the divalent anions SO and oxalate by Slc26a1 is strongly activated by impermeant, monovalent anions such as Cl− and formate. This is, presumably, not simply a matter of the valence of anionic charge, because HCO reportedly does not activate SO uptake by rat Slc26a1 (43). The physiological significance of this phenomenon is not yet clear; however, this functional characteristic of Slc26a1 is dramatically different from that of other SLC26 anion exchangers, including Slc26a2 (43). It is hoped that a comparative structure-function analysis of Slc26a1 and Slc26a2 will yield some insight into the molecular mechanism. As shown in Fig. 4, SO exchange by Slc26a1 is significantly stimulated by an acid-outside pH gradient, particularly in the presence of extracellular Cl− (Fig. 4 C). This is suggestive of H+-SO cotransport, as reported for AE1 (reviewed in Ref. 6), a member of the HCO transporter superfamily (SLC4) that can also mediate the transport of SO , Cl−, and HCO .
The finding that Slc26a6 functions in the exchange of formate and oxalate for both Cl− and SO is of particular relevance to its role in the kidney. In conjunction with Na+/H+ exchange mediated by NHE3, apical Cl−/formate exchange functions in transepithelial reabsorption of NaCl by the proximal tubule (57) and some segments of the distal nephron (55). The perfusion of proximal tubules with luminal oxalate also stimulates transepithelial salt transport, although in this case Na+-SO cotransport rather than Na+/H+ exchange absorbs Na+(see below) (56). An important observation is that Slc26a6 mediates both Cl−/formate and Cl−/oxalate exchange, given that previous studies using renal brush-border vesicles suggested the existence of two separate distinct transporters, one capable of only Cl−/formate exchange and the other capable of both Cl−/formate and Cl−/oxalate exchange (16). The Cl−/formate/oxalate exchanger activity in brush-border vesicles has a higher affinity for oxalate over formate (16); although kinetic studies of Slc26a6 are lacking, the greater efficacy of oxalate in bothcis-inhibition of 36Cl− uptake (Fig. 5 A) and trans-stimulation of35SO efflux (Fig. 5 C) are consistent with this property. Unlike Cl−/formate exchange, Cl−/oxalate exchange in apical membrane vesicles is thought to be electrogenic (16), compatible perhaps with the observation that Cl−/HCO exchange by Slc26a6 is not electroneutral (Fig. 8). The question of whether two separate gene products mediate Cl−/formate exchange in the proximal tubule awaits the characterization of Slc26a6 knockout mice, along with the full pharmacological characterization of the Slc26a6 in its various transport modes (16). A second unresolved issue is how the luminal exchange of Cl− with formate and oxalate is specifically coupled to Na+/H+ exchange and Na+-SO cotransport, respectively (56). This lack of physiological coupling between luminal formate and apical Na+-SO cotransport remains unexplained, because Slc26a6 can evidently mediate both SO /formate and Cl−/formate exchange (Fig. 5).
Finally, another longstanding issue in the transcellular transport of NaCl by the proximal tubule has been the relative importance of apical Cl−/formate/oxalate exchange and Cl−/base exchange, particularly Cl−/OH− exchange. Whereas several groups have reported the presence of apical Cl−/base exchange in both vesicle and whole tubule preparations, this has not been reproduced in many other studies (reviewed in Ref. 24). Data shown in Fig. 9 indicate that Slc26a6 can clearly mediate Cl−/OH− exchange. Again, clarification of the quantitative role of Slc26a6 in apical Cl−/OH− exchange in the proximal tubule awaits the characterization of Slc26a6-null mice.
The expression pattern and substrate specificity of Slc26a6 suggests that it may mediate several modes of anion exchange in a number of tissues. For example, there is evidence for Cl−/formate exchange in vascular smooth muscle and cardiac myocytes (50), consistent with the expression of Slc26a6 in heart and skeletal muscle (Fig. 2). The role of Slc26a6 in Cl−/HCO exchange (Fig. 8) is of particular relevance to the physiology of tissues that excrete HCO under the influence of CFTR (7, 26,27). Suggestive evidence that CFTR might regulate SLC26A6 was initially reported in a study of cystic fibrosis pancreatic cell lines, in which the expression of wild-type CFTR results in a 10-fold activation of DIDS-sensitive sulfate transport (8). More recently, Greeley et al. (12) reported that stable transfection of cystic fibrosis pancreatic cell lines with wild-type CFTR results in a significant stimulation of Cl−/HCO exchange, along with the induction of transcripts encoding both SLC26A3 and SLC26A6 (12). Additionally, a correlation was recently made between the ability of CFTR mutants to regulate Cl−/HCO exchange and the associated cystic fibrosis phenotype; whereas mutants associated with pancreatic insufficiency do not promote cAMP-dependent Cl−/HCO exchange, this regulation is preserved in mutants associated with pancreatic sufficiency (7).
Finally, the ability of Slc26a1 and Slc26a6 to mediate oxalate exchange suggests important roles for these transporters in oxalate homeostasis. Thus there is evidence for DIDS-sensitive Cl−/oxalate, OH−/oxalate, and formate/oxalate exchange in brush-border vesicles from rabbit ileum (20), and Slc26a6 is heavily expressed in small intestine (Fig. 2). Slc26a1 and Slc26a6 are expressed at the basolateral (17) and apical (19) membranes, respectively, of the proximal tubule, a major site of renal oxalate secretion (47). Hyperexcretion of oxalate is an important factor in the pathogenesis of renal stones, and increased red cell oxalate transport has been shown to segregate with oxalate excretion in certain kindreds with nephrolithiasis (3); although red cell oxalate transport has generally been attributed to AE1 (15), this has not to our knowledge been verified by heterologous expression, and the relative role of specific anion exchangers in oxalate transport is as yet unknown. However, it is evident that dietary absorption of oxalate, potentially via SLC26A6, is an important determinant of urinary excretion (14). Given this physiology, variation in the humanSLC26A1 and/or SLC26A6 genes may be an important determinant of the risk for nephrolithiasis.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants K11-DK-02103 (D. B. Mount), PO1-DK-038226 (D. B. Mount), RO1-DK-57708 (D. B. Mount), and RO1-DK-56218 (M. F. Romero) and by a Howard Hughes Medical Institute grant to Case Western Reserve University (M. F. Romero). D. B. Mount is supported by an Advanced Career Development Award from the Department of Veterans Affairs; Q. Xie was supported by NIDDK training grant T32-DK-07569–12.
↵* Q. Xie and R. Welch contributed equally to this study.
Address for reprint requests and other correspondence: D. B. Mount, Dept. of Veterans Affairs Medical Center, 1400 VFW Pkwy., West Roxbury, MA, 02132 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajprenal.00079.2002
- Copyright © 2002 the American Physiological Society